Nanopore-based sequencing using voltage mode with hybrid mode stimuli

ABSTRACT

A liquid voltage is applied to a first side of a lipid bilayer. The liquid voltage comprises a tag-reading period with a tag-reading voltage that tends to capture a tag into a nanopore in the lipid bilayer and an open-channel period with an open-channel voltage that tends to repel the tag. A pre-charging voltage source is connected to an integrating capacitor and a working electrode on a second side of the lipid bilayer during a pre-charging time period, such that the integrating capacitor and the working electrode are charged to a pre-charging voltage. The pre-charging voltage source is disconnected from the integrating capacitor and the working electrode during an integrating time period, such that a voltage of the integrating capacitor and a voltage of the working electrode may vary as a current flows through the nanopore. The pre-charging time period overlaps with a beginning portion of the tag-reading period.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Patent ApplicationNo. PCT/EP2017/073095, filed Sep. 14, 2017, which claims priority toU.S. Provisional Application No. 62/394,903, filed Sep. 15, 2016, eachof which is herein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Advances in micro-miniaturization within the semiconductor industry inrecent years have enabled biotechnologists to begin packingtraditionally bulky sensing tools into smaller and smaller form factors,onto so-called biochips. It would be desirable to develop techniques forbiochips that make them more robust, efficient, and cost-effective.

BRIEF DESCRIPTION Brief Description of the Drawings

Various embodiments of the invention are disclosed in the followingdetailed description and the accompanying drawings.

FIG. 1 illustrates an embodiment of a cell 100 in a nanopore basedsequencing chip.

FIG. 2 illustrates an embodiment of a cell 200 performing nucleotidesequencing with the Nano-SBS technique.

FIG. 3 illustrates an embodiment of a cell about to perform nucleotidesequencing with pre-loaded tags.

FIG. 4 illustrates an embodiment of a process 400 for nucleic acidsequencing with pre-loaded tags.

FIG. 5 illustrates an embodiment of a cell 500 in a nanopore basedsequencing chip.

FIG. 6 illustrates an embodiment of a circuitry 600 in a cell of ananopore based sequencing chip.

FIG. 7A illustrates an embodiment of a circuitry 700 in a cell of ananopore based sequencing chip, wherein the voltage applied across thenanopore can be configured to vary over a time period during which thenanopore is in a particular detectable state.

FIG. 7B illustrates an additional embodiment of a circuitry 700 in acell of a nanopore based sequencing chip, wherein the voltage appliedacross the nanopore can be configured to vary over a time period duringwhich the nanopore is in a particular detectable state.

FIG. 8 illustrates an embodiment of a process 800 for analyzing amolecule inside a nanopore, wherein the nanopore is inserted in amembrane.

FIG. 9 illustrates an embodiment of a plot of the voltage at theintegrating capacitor (V_(ncap)) versus time when process 800 isperformed and repeated three times within a bright period of the ACvoltage source signal cycle.

FIG. 10 illustrates an embodiment of the plots of the voltage at theintegrating capacitor versus time when the nanopore is in differentstates.

FIG. 11 illustrates an embodiment of a time signal plot of a pluralityof signals in a cell of the nanopore based sequencing chip.

FIG. 12 illustrates another embodiment of a reset signal that is used tocontrol the switch that connects or disconnects the voltage source to orfrom the membrane in a cell of the nanopore based sequencing chip.

FIG. 13 illustrates another embodiment of a reset signal that is used tocontrol the switch that connects or disconnects the voltage source to orfrom the membrane in a cell of the nanopore based sequencing chip.

FIG. 14 illustrates yet another embodiment of a reset signal that isused to control the switch that connects or disconnects the voltagesource to or from the membrane in a cell of the nanopore basedsequencing chip.

DETAILED DESCRIPTION

The invention can be implemented in numerous ways, including as aprocess; an apparatus; a system; a composition of matter; a computerprogram product embodied on a computer readable storage medium; and/or aprocessor, such as a processor configured to execute instructions storedon and/or provided by a memory coupled to the processor. In thisspecification, these implementations, or any other form that theinvention may take, may be referred to as techniques. In general, theorder of the steps of disclosed processes may be altered within thescope of the invention. Unless stated otherwise, a component such as aprocessor or a memory described as being configured to perform a taskmay be implemented as a general component that is temporarily configuredto perform the task at a given time or a specific component that ismanufactured to perform the task. As used herein, the term ‘processor’refers to one or more devices, circuits, and/or processing coresconfigured to process data, such as computer program instructions.

A detailed description of one or more embodiments of the invention isprovided below along with accompanying figures that illustrate theprinciples of the invention. The invention is described in connectionwith such embodiments, but the invention is not limited to anyembodiment. The scope of the invention is limited only by the claims andthe invention encompasses numerous alternatives, modifications andequivalents. Numerous specific details are set forth in the followingdescription in order to provide a thorough understanding of theinvention. These details are provided for the purpose of example and theinvention may be practiced according to the claims without some or allof these specific details. For the purpose of clarity, technicalmaterial that is known in the technical fields related to the inventionhas not been described in detail so that the invention is notunnecessarily obscured.

Nanopore membrane devices having pore sizes on the order of onenanometer in internal diameter have shown promise in rapid nucleotidesequencing. When a voltage potential is applied across a nanoporeimmersed in a conducting fluid, a small ion current attributed to theconduction of ions through the nanopore can be observed. The size of thecurrent is sensitive to the pore size.

A nanopore based sequencing chip may be used for nucleic acid (e.g.,DNA) sequencing. A nanopore based sequencing chip incorporates a largenumber of sensor cells configured as an array. For example, an array ofone million cells may include 1000 rows by 1000 columns of cells.

FIG. 1 illustrates an embodiment of a cell 100 in a nanopore basedsequencing chip. A membrane 102 is formed over the surface of the cell.In some embodiments, membrane 102 is a lipid bilayer. The bulkelectrolyte 114 containing soluble protein nanopore transmembranemolecular complexes (PNTMC) and the analyte of interest is placeddirectly onto the surface of the cell. In one embodiment, a single PNTMC104 is inserted into membrane 102 by electroporation. The individualmembranes in the array are neither chemically nor electrically connectedto each other. Thus, each cell in the array is an independent sequencingmachine, producing data unique to the single polymer molecule associatedwith the PNTMC. PNTMC 104 operates on the analytes and modulates theionic current through the otherwise impermeable bilayer.

With continued reference to FIG. 1, analog measurement circuitry 112 isconnected to a metal electrode 110 covered by a volume of electrolyte108. The volume of electrolyte 108 is isolated from the bulk electrolyte114 by the ion-impermeable membrane 102. PNTMC 104 crosses membrane 102and provides the only path for ionic current to flow from the bulkliquid to working electrode 110. The cell also includes a counterelectrode (CE) 116, which is in electrical contact with the bulkelectrolyte 114. The cell may also include a reference electrode 117.

In some embodiments, a nanopore array enables parallel sequencing usingthe single molecule nanopore-based sequencing by synthesis (Nano-SBS)technique. FIG. 2 illustrates an embodiment of a cell 200 performingnucleotide sequencing with the Nano-SBS technique. In the Nano-SBStechnique, a template 202 to be sequenced and a primer are introduced tocell 200. To this template-primer complex, four differently taggednucleotides 208 are added to the bulk aqueous phase. As the correctlytagged nucleotide is complexed with the polymerase 204, the tail of thetag is positioned in the barrel of nanopore 206. The tag held in thebarrel of nanopore 206 generates a unique ionic blockade signal 210,thereby electronically identifying the added base due to the tags'distinct chemical structures.

FIG. 3 illustrates an embodiment of a cell about to perform nucleotidesequencing with pre-loaded tags. A nanopore 301 is formed in a membrane302. An enzyme 303 (e.g., a polymerase, such as a DNA polymerase) isassociated with the nanopore. In some cases, polymerase 303 iscovalently attached to nanopore 301. Polymerase 303 is associated with anucleic acid molecule 304 to be sequenced. In some embodiments, thenucleic acid molecule 304 is circular. In some cases, nucleic acidmolecule 304 is linear. In some embodiments, a nucleic acid primer 305is hybridized to a portion of nucleic acid molecule 304. Polymerase 303catalyzes the incorporation of nucleotides 306 onto primer 305 usingsingle stranded nucleic acid molecule 304 as a template. Nucleotides 306comprise tag species (“tags”) 307.

FIG. 4 illustrates an embodiment of a process 400 for nucleic acidsequencing with pre-loaded tags. Stage A illustrates the components asdescribed in FIG. 3. Stage C shows the tag loaded into the nanopore. A“loaded” tag may be one that is positioned in and/or remains in or nearthe nanopore for an appreciable amount of time, e.g., 0.1 millisecond(ms) to 10,000 ms. In some cases, a tag that is pre-loaded is loaded inthe nanopore prior to being released from the nucleotide. In someinstances, a tag is pre-loaded if the probability of the tag passingthrough (and/or being detected by) the nanopore after being releasedupon a nucleotide incorporation event is suitably high, e.g., 90% to99%.

At stage A, a tagged nucleotide (one of four different types: A, T, G,or C) is not associated with the polymerase. At stage B, a taggednucleotide is associated with the polymerase. At stage C, the polymeraseis docked to the nanopore. The tag is pulled into the nanopore duringdocking by an electrical force, such as a force generated in thepresence of an electric field generated by a voltage applied across themembrane and/or the nanopore.

Some of the associated tagged nucleotides are not base paired with thenucleic acid molecule. These non-paired nucleotides typically arerejected by the polymerase within a time scale that is shorter than thetime scale for which correctly paired nucleotides remain associated withthe polymerase. Since the non-paired nucleotides are only transientlyassociated with the polymerase, process 400 as shown in FIG. 4 typicallydoes not proceed beyond stage D. For example, a non-paired nucleotide isrejected by the polymerase at stage B or shortly after the processenters stage C.

Before the polymerase is docked to the nanopore, the conductance of thenanopore is ˜300 picosiemens (300 pS). At stage C, the conductance ofthe nanopore is about 60 pS, 80 pS, 100 pS, or 120 pS, corresponding toone of the four types of tagged nucleotides respectively. The polymeraseundergoes an isomerization and a transphosphorylation reaction toincorporate the nucleotide into the growing nucleic acid molecule andrelease the tag molecule. In particular, as the tag is held in thenanopore, a unique conductance signal (e.g., see signal 210 in FIG. 2)is generated due to the tag's distinct chemical structures, therebyidentifying the added base electronically. Repeating the cycle (i.e.,stage A through E or stage A through F) allows for the sequencing of thenucleic acid molecule. At stage D, the released tag passes through thenanopore.

In some cases, tagged nucleotides that are not incorporated into thegrowing nucleic acid molecule will also pass through the nanopore, asseen in stage F of FIG. 4. The unincorporated nucleotide can be detectedby the nanopore in some instances, but the method provides a means fordistinguishing between an incorporated nucleotide and an unincorporatednucleotide based at least in part on the time for which the nucleotideis detected in the nanopore. Tags bound to unincorporated nucleotidespass through the nanopore quickly and are detected for a short period oftime (e.g., less than 10 ms), while tags bound to incorporatednucleotides are loaded into the nanopore and detected for a long periodof time (e.g., at least 10 ms).

FIG. 5 illustrates an embodiment of a cell 500 in a nanopore basedsequencing chip. Cell 500 includes a well 505 having two side walls anda bottom. In one embodiment, each side wall comprises a dielectric layer504 and the bottom comprises a working electrode 502. In one embodiment,the working electrode 502 has a top side and a bottom side. In anotherembodiment, the top side of 502 makes up the bottom of the well 505while the bottom side of 502 is in contact with dielectric layer 501. Inanother embodiment, the dielectric layer 504 is above dielectric layer501. Dielectric layer 504 forms the walls surrounding a well 505 inwhich a working electrode 502 is located at the bottom. Suitabledielectric materials for use in the present invention (e.g., dielectriclayer 501 or 504) include, without limitation, porcelain (ceramic),glass, mica, plastics, oxides, nitrides (e.g., silicon mononitride orSiN), silicon oxynitride, metal oxides, metal nitrides, metal silicates,transition-metal oxides, transition-metal nitrides, transitionmetal-silicates, oxynitrides of metals, metal aluminates, zirconiumsilicate, zirconium aluminate, hafnium oxide, insulating materials(e.g., polymers, epoxies, photoresist, and the like), or combinationsthereof. Those of ordinary skill in the art will appreciate otherdielectric materials that are suitable for use in the present invention.

In one aspect, cell 500 also includes one or more hydrophobic layers. Asshown in FIG. 5, each dielectric layer 504 has a top surface. In oneembodiment, the top surface of each dielectric layer 504 may comprise ahydrophobic layer. In one embodiment, silanization forms a hydrophobiclayer 520 above the top surface of dielectric layer 504. For example,further silanization with silane molecules (i) containing 6 to 20carbon-long chains (e.g., octadecyl-trichlorosilane,octadecyl-trimethoxysilane, or octadecyl-triethoxysilane), (ii)dimethyloctylchlorosilane (DMOC), or (iii) organofunctional alkoxysilanemolecules (e.g., dimethylchloro-octodecyl-silane,methyldichloro-octodecyl-silane, trichloro-octodecyl-silane,trimethyl-octodecyl-silane, or triethyl-octodecyl-silane) can be done onthe top surface of dielectric layer 504. In one embodiment, thehydrophobic layer is a silanized layer or silane layer. In oneembodiment, the silane layer can be one molecule in thickness. In oneaspect, dielectric layer 504 comprises a top surface suitable foradhesion of a membrane (e.g., a lipid bilayer comprising a nanopore). Inone embodiment, the top surface suitable for adhesion of a membranecomprises a silane molecule as described herein. In some embodiments,hydrophobic layer 520 has a thickness provided in a nanometer (nM) ormicrometer (μm) scale. In other embodiments, the hydrophobic layer mayextend down along all or a part of the dielectric layer 504. (see alsoDavis et al. U.S. 20140034497, which is incorporated herein by referencein its entirety).

In another aspect, well 505 (formed by the dielectric layer walls 504)further includes a volume of salt solution 506 above working electrode502. In general, the methods of the present invention comprise the useof a solution (e.g., a salt solution, salt buffer solution, electrolyte,electrolyte solution, or bulk electrolyte) that comprises osmolytes. Asused herein, the term “osmolyte” refers to any soluble compound thatwhen dissolved into solution increases the osmolarity of that solution.In the present invention, an osmolyte is a compound that is soluble insolution within the architecture of a nanopore sequencing system, e.g.,a well containing a salt solution or a bulk electrolyte as describedherein. As such, the osmolytes of the present invention affect osmosis,particularly osmosis across a lipid bilayer. Osmolytes for use in thepresent invention include, without limitation, ionic salts such aslithium chloride (LiCl), sodium chloride (NaCl), potassium chloride(KCl), lithium glutamate, sodium glutamate, potassium glutamate, lithiumacetate, sodium acetate, potassium acetate, calcium chloride (CaCl₂),strontium chloride (SrCl₂), manganese chloride (MnCl₂), and magnesiumchloride (MgCl₂); polyols and sugars such as glycerol, erythritol,arabitol, sorbitol, mannitol, xylitol, mannisidomannitol, glycosylglycerol, glucose, fructose, sucrose, trehalose, and isofluoroside;polymers such as dextrans, levans, and polyethylene glycol; and someamino acids and derivatives thereof such as glycine, alanine,alpha-alanine, arginine, proline, taurine, betaine, octopine, glutamate,sarcosine, y-aminobutyric acid, and trimethylamine N-oxide (“TMAO”) (seealso e.g., Fisher et al. U.S. 20110053795, incorporated herein byreference in its entirety). In one embodiment, the present inventionutilizes a solution comprising an osmolyte, wherein the osmolyte is anionic salt. Those of ordinary skill in the art will appreciate othercompounds that are suitable osmolytes for use in the present invention.In another aspect, the present invention provides solutions comprisingtwo or more different osmolytes.

The architecture of the nanopore based sequencing chip described hereincomprises an array of wells (e.g., FIG. 5) having a volume of between 1attoliter and 1 nanoliter.

As shown in FIG. 5, a membrane is formed on the top surfaces ofdielectric layer 504 and spans across well 505. For example, themembrane includes a lipid monolayer 518 formed on top of hydrophobiclayer 520. As the membrane reaches the opening of well 505, the lipidmonolayer transitions to a lipid bilayer 514 that spans across theopening of the well. The lipid monolayer 518 may also extend along allor a part of the vertical surface (i.e., side wall) of a dielectriclayer 504. In one embodiment, the vertical surface 504 along which themonolayer 518 extends comprises a hydrophobic layer. A bulk electrolyte508 containing protein nanopore transmembrane molecular complexes(PNTMC) and the analyte of interest is placed directly above the well. Asingle PNTMC/nanopore 516 is inserted into lipid bilayer 514. In oneembodiment, insertion into the bilayer is by electroporation. Nanopore516 crosses lipid bilayer 514 and provides the only path for ionic flowfrom bulk electrolyte 508 to working electrode 502.

Cell 500 includes a counter electrode (CE) 510, which is in electricalcontact with the bulk electrolyte 508. Cell 500 may optionally include areference electrode 512. In some embodiments, counter electrode 510 isshared between a plurality of cells, and is therefore also referred toas a common electrode. The common electrode can be configured to apply acommon potential to the bulk liquid in contact with the nanopores in themeasurements cells. The common potential and the common electrode arecommon to all of the measurement cells.

In some embodiments, working electrode 502 is a metal electrode. Fornon-faradaic conduction, working electrode 502 may be made of metalsthat are resistant to corrosion and oxidation, e.g., platinum, gold,titanium nitride and graphite. For example, working electrode 502 may bea platinum electrode with electroplated platinum. In another example,working electrode 502 may be a titanium nitride (TiN) working electrode.

As shown in FIG. 5, nanopore 516 is inserted into the planar lipidbilayer 514 suspended over well 505. An electrolyte solution is presentboth inside well 505, i.e., trans side, (see salt solution 506) and in amuch larger external reservoir 522, i.e., cis side, (see bulkelectrolyte 508). The bulk electrolyte 508 in external reservoir 522 isabove multiple wells of the nanopore based sequencing chip. Lipidbilayer 514 extends over well 505 and transitions to lipid monolayer 518where the monolayer is attached to hydrophobic layer 520. This geometryboth electrically and physically seals well 505 and separates the wellfrom the larger external reservoir. While neutral molecules, such aswater and dissolved gases, may pass through lipid bilayer 514, ions maynot. Nanopore 516 in lipid bilayer 514 provides a single path for ionsto be conducted into and out of well 505.

For nucleic acid sequencing, a polymerase is attached to nanopore 516. Atemplate of nucleic acid (e.g., DNA) is held by the polymerase. Forexample, the polymerase synthesizes DNA by incorporating hexaphosphatemono-nucleotides (HMN) from solution that are complementary to thetemplate. A unique, polymeric tag is attached to each HMN. Duringincorporation, the tag threads the nanopore aided by an electric fieldgradient produced by the voltage between counter electrode 510 andworking electrode 502. The tag partially blocks nanopore 516, procuringa measurable change in the ionic current through nanopore 516. In someembodiments, an alternating current (AC) bias or direct current (DC)voltage is applied between the electrodes.

FIG. 6 illustrates an embodiment of a circuitry 600 in a cell of ananopore based sequencing chip. As mentioned above, when the tag is heldin nanopore 602, a unique conductance signal (e.g., see signal 210 inFIG. 2) is generated due to the tag's distinct chemical structures,thereby identifying the added base electronically. The circuitry in FIG.6 maintains a constant voltage across nanopore 602 when the current flowis measured. In particular, the circuitry includes an operationalamplifier 604 and a pass device 606 that maintain a constant voltageequal to V_(a) or V_(b) across nanopore 602. The current flowing throughnanopore 602 is integrated at a capacitor n_(cap) 608 and measured by anAnalog-to-Digital (ADC) converter 610.

However, circuitry 600 has a number of drawbacks. One of the drawbacksis that circuitry 600 only measures unidirectional current flow. Anotherdrawback is that operational amplifier 604 in circuitry 600 mayintroduce a number of performance issues. For example, the offsetvoltage and the temperature drift of operational amplifier 604 may causethe actual voltage applied across nanopore 602 to vary across differentcells. The actual voltage applied across nanopore 602 may drift by tensof millivolts above or below the desired value, thereby causingsignificant measurement inaccuracies. In addition, the operationalamplifier noise may cause additional detection errors. Another drawbackis that the portions of the circuitry for maintaining a constant voltageacross the nanopore while current flow measurements are made arearea-intensive. For example, operational amplifier 604 occupiessignificantly more space in a cell than other components. As thenanopore based sequencing chip is scaled to include more and more cells,the area occupied by the operational amplifiers may increase to anunattainable size. Unfortunately, shrinking the operational amplifier'ssize in a nanopore based sequencing chip with a large-sized array mayraise other performance issues; for example, it may exacerbate theoffset and noise problems in the cells even further. Therefore, animproved circuitry and an improved method to analyze molecules usingnanopores would be desirable.

FIG. 7A illustrates an embodiment of a circuitry 700 in a cell of ananopore based sequencing chip, wherein the voltage applied across thenanopore can be configured to vary over a time period during which thenanopore is in a particular detectable state. One of the possible statesof the nanopore is an open-channel state, in which a tag-attachedpolyphosphate is absent from the barrel of the nanopore. Another fourpossible states of the nanopore correspond to the states when the fourdifferent types of tag-attached polyphosphate (A, T, G, or C) are heldin the barrel of the nanopore. Yet another possible state of thenanopore is when the membrane is ruptured. In circuitry 700, theoperational amplifier is no longer required.

FIG. 7A shows a nanopore 702 that is inserted into a membrane 712, andnanopore 702 and membrane 712 are situated between a cell workingelectrode 714 and a counter electrode 716, such that a voltage isapplied across nanopore 702. Nanopore 702 is also in contact with a bulkliquid/electrolyte 718. Note that working electrode 714, nanopore 702,membrane 712, and counter electrode 716 are drawn upside down ascompared to the working electrode, nanopore, membrane, and counterelectrode in FIG. 1. Hereinafter, a cell is meant to include at least amembrane, a nanopore, a working cell electrode, and the associatedcircuitry. In some embodiments, the counter electrode is shared betweena plurality of cells, and is therefore also referred to as a commonelectrode. The common electrode can be configured to apply a commonpotential to the bulk liquid in contact with the nanopores in themeasurements cells by connecting the common electrode to a voltagesource V_(liq) 720. The common potential and the common electrode arecommon to all of the measurement cells. There is a working cellelectrode within each measurement cell; in contrast to the commonelectrode, working cell electrode 714 is configurable to apply adistinct potential that is independent from the working cell electrodesin other measurement cells.

FIG. 7B illustrates the same circuitry 700 in a cell of a nanopore basedsequencing chip as that shown in FIG. 7A. Comparing to FIG. 7A, insteadof showing a nanopore inserted in a membrane between the workingelectrode and the counter electrode, an electrical model 722representing the electrical properties of the nanopore and membrane isshown.

Electrical model 722 includes a capacitor 726 (C_(bilayer)) that modelsa capacitance associated with the lipid bilayer and a resistor 728(R_(bilayer)) that models a resistance associated with thenanopore/lipid bilayer in different states (e.g., the open-channel stateor the states corresponding to having different types of tag/moleculeinside the nanopore).

Voltage source V_(liq) 720 is an alternating current (AC) voltagesource. Counter electrode 716 is immersed in the bulk liquid 718, and anAC non-Faradaic mode is utilized to modulate a square wave voltageV_(liq) and apply it to the bulk liquid in contact with the lipidmembranes/bilayers in the measurement cells. In some embodiments,V_(liq) is a square wave with a peak-to-peak magnitude variation of100-250 mV and a frequency between 20 and 400 Hz.

Pass device 706 is a switch that can be used to connect or disconnectthe lipid membrane/bilayer and the electrodes from the measurementcircuitry 700. The switch enables or disables a voltage stimulus thatcan be applied across the lipid membrane/bilayer in the cell. Beforelipids are deposited to the cell to form a lipid bilayer, the impedancebetween the two electrodes is very low because the well of the cell isnot sealed, and therefore switch 706 is kept open to avoid ashort-circuit condition. Switch 706 may be closed once lipid solvent hasbeen deposited to the cell that seals the well of the cell.

Circuitry 700 further includes an on-chip fabricated integratingcapacitor 708 (n_(cap)). Integrating capacitor 708 is pre-charged byusing a reset signal 703 to close switch 701, such that integratingcapacitor 708 is connected to a voltage source V_(pre) 705. In someembodiments, voltage source V_(pre) 705 provides a constant positivevoltage with a magnitude of 900 mV. When switch 701 is closed,integrating capacitor 708 is pre-charged to the positive voltage levelof voltage source V_(pre) 705.

After integrating capacitor 708 is pre-charged, reset signal 703 is usedto open switch 701 such that integrating capacitor 708 is disconnectedfrom voltage source V_(pre) 705. At this point, depending on the levelof V_(liq), the potential of counter electrode 716 may be at a higherlevel than the potential of working electrode 714, or vice versa. Forexample, during the positive phase of square wave V_(liq) (i.e., thedark period of the AC voltage source signal cycle), the potential ofcounter electrode 716 is at a higher level than the potential of workingelectrode 714. Similarly, during the negative phase of square waveV_(liq) (i.e., the bright period of the AC voltage source signal cycle),the potential of counter electrode 716 is at a lower level than thepotential of working electrode 714. Due to this potential difference,integrating capacitor 708 may be charged during the dark period of theAC voltage source signal cycle and discharged during the bright periodof the AC voltage source signal cycle. The charging or dischargingcauses an ionic current to flow through the nanopore.

Integrating capacitor 708 charges or discharges during a time periodwhen switch 701 is opened by reset signal 703, and at the end of thistime period, the voltage stored in integrating capacitor 708 may be readout by ADC 710. After the sampling by ADC 710, integrating capacitor 708may be pre-charged again by using reset signal 703 to close switch 701,such that integrating capacitor 708 is connected to voltage sourceV_(pre) 705 again. In some embodiments, the sampling rate of ADC 710 isbetween 500 to 2000 Hz. In some embodiments, the sampling rate of ADC710 is up to 5 kHz. For example, with a sampling rate of 1 kHz,integrating capacitor 708 may be charged or discharged for a period of˜1 ms, and then the voltage at integrating capacitor 708 is read out byADC 710. After the sampling by ADC 710, integrating capacitor 708 ispre-charged again by using reset signal 703 to close switch 701 suchthat integrating capacitor 708 is connected to voltage source V_(pre)705 again. The steps of pre-charging the integrating capacitor 708,waiting a fixed period of time for the integrating capacitor 708 tocharge or discharge, and sampling the voltage stored in integratingcapacitor by ADC 710 are then repeated in cycles. As will be describedin greater detail below, the rate of voltage decay of the integratingcapacitor caused by the charging and discharging of the capacitor may bemonitored and used to analyze a molecule inside a nanopore.

FIG. 8 illustrates an embodiment of a process 800 for analyzing amolecule inside a nanopore, wherein the nanopore is inserted in amembrane. Process 800 may be performed using the circuitries shown inFIGS. 7A and 7B. FIG. 9 illustrates an embodiment of a plot of thevoltage at the integrating capacitor (V_(ncap)) versus time when process800 is performed and repeated three times within a bright period of theAC voltage source signal cycle. Due to the discharging of theintegrating capacitor, the voltage applied across the nanopore is notheld constant. Instead, the voltage applied across the nanopore changesover time. The rate of the voltage decay (i.e., the steepness of theslope of the voltage at the integrating capacitor versus time plot)depends on the cell resistance (e.g., the resistance of resistor 728 inFIG. 7B). More particularly, as the resistance associated with thenanopore in different states (e.g., the open-channel state, the statescorresponding to having different types of tag/molecule inside thenanopore, and the state when the membrane is ruptured) are different dueto the molecules'/tags' distinct chemical structures, differentcorresponding rates of voltage decay may be observed and thus may beused to identify the different states of the nanopore.

With reference to FIG. 8 and FIG. 7B, at 802 of process 800, theintegrating capacitor is pre-charged by coupling the integratingcapacitor and the nanopore to a pre-charging voltage source. Forexample, as shown in FIG. 7B, integrating capacitor 708 is pre-chargedto V_(pre) by using a reset signal 703 to close switch 701, such thatintegrating capacitor 708 and the nanopore are coupled to a voltagesource V_(pre) 705. As shown in FIG. 9, the initial voltage atintegrating capacitor is V_(pre). As switch 701 is closed and theintegrating capacitor is being pre-charged, the capacitor associatedwith the membrane is also charged simultaneously and energy is stored inan electric field across the membrane.

At 804 of process 800, the capacitor associated with the membrane(capacitor 726) and the integrating capacitor are discharged bydecoupling the nanopore and the membrane from the voltage source, andthe energy stored in the electric field across the membrane is therebydissipated. For example, as shown in FIG. 7B, the voltage source V_(pre)705 is disconnected when switch 701 is opened by a reset signal 703.After switch 701 is opened, the voltage at the integrating capacitorbegins to decay exponentially, as shown in FIG. 9. The exponential decayhas a RC time constant τ=RC, where R is the resistance associated withthe nanopore (resistor 728) and C is the capacitance in parallel with R,including the capacitance associated with the membrane C_(bilayer) 726and the capacitance associated with n_(cap) 708.

At 806 of process 800, a rate of decay of the voltage at the integratingcapacitor is determined. The rate of voltage decay is the steepness ofthe slope of the voltage at the integrating capacitor versus time plot,as shown in FIG. 9. The rate of voltage decay may be determined indifferent ways.

In some embodiments, the rate of voltage decay is determined bymeasuring the voltage decay that occurs during a fixed time interval.For example, as shown in FIG. 9, the voltage at the integratingcapacitor is first measured by ADC 710 at time t₁, and then the voltageis again measured by ADC 710 at time t₂. The voltage difference ΔV isgreater when the slope of the V_(ncap) voltage versus time curve issteeper, and the voltage difference ΔV is smaller when the slope of thevoltage versus time curve is less steep. Thus, ΔV may be used as ametric for determining the rate of decay of the voltage at theintegrating capacitor. In some embodiments, to increase the accuracy ofthe measurement of the rate of voltage decay, the voltage may bemeasured additional times at fixed intervals. For example, the voltagemay be measured at times t₃, t₄, and so on, and the multiplemeasurements of ΔV during the multiple time intervals may be jointlyused as a metric for determining the rate of decay of the voltage at theintegrating capacitor. In some embodiments, correlated double sampling(CDS) may be used to increase the accuracy of the measurement of therate of voltage decay.

In some embodiments, the rate of voltage decay is determined bymeasuring the time duration that is required for a selected amount ofvoltage decay. In some embodiments, the time required for the voltage todrop from a fixed voltage V₁ to a second fixed voltage V₂ may bemeasured. The time required is less when the slope of the voltage versustime curve is steeper, and the time required is greater when the slopeof the voltage versus time curve is less steep. Thus, the measured timerequired may be used as a metric for determining the rate of decay ofthe voltage at the integrating capacitor.

At 808 of process 800, a state of the nanopore is determined based onthe determined rate of voltage decay. One of the possible states of thenanopore is an open-channel state during which a tag-attachedpolyphosphate is absent from the barrel of the nanopore. Other possiblestates of the nanopore correspond to the states when different types ofmolecules are held in the barrel of the nanopore. For example, anotherfour possible states of the nanopore correspond to the states when thefour different types of tag-attached polyphosphate (A, T, G, or C) areheld in the barrel of the nanopore. Yet another possible state of thenanopore is when the membrane is ruptured. The state of the nanopore canbe determined based on the determined rate of voltage decay, because therate of voltage decay depends on the cell resistance; i.e., theresistance of resistor 728 in FIG. 7B. More particularly, as theresistances associated with the nanopore in different states aredifferent due to the molecules/tags' distinct chemical structures,different corresponding rates of voltage decay may be observed and thusmay be used to identify the different states of the nanopore.

FIG. 10 illustrates an embodiment of the plots of the voltage at theintegrating capacitor versus time when the nanopore is in differentstates. Plot 1002 shows the rate of voltage decay during an open-channelstate. In some embodiments, the resistance associated with the nanoporein an open-channel state is in the range of 100 Mohm to 20 Gohm. Plots1004, 1006, 1008, and 1010 show the different rates of voltage decaycorresponding to the four capture states when the four different typesof tag-attached polyphosphate (A, T, G, or C) are held in the barrel ofthe nanopore. In some embodiments, the resistance associated with thenanopore in a capture state is within the range of 200 Mohm to 40 Gohm.Note that the slope of each of the plots is distinguishable from eachother.

At 810 of process 800, it is determined whether process 800 is repeated.For example, the process may be repeated a plurality of times to detecteach state of the nanopore. If the process is not repeated, then process800 terminates; otherwise, the process restarts at 802 again. At 802,the integrating capacitor is pre-charged again by coupling theintegrating capacitor and the nanopore to the pre-charging voltagesource. For example, as shown in FIG. 7B, integrating capacitor 708 ispre-charged to V_(pre) by using reset signal 703 to close switch 701,such that integrating capacitor 708 and the nanopore are coupled to avoltage source V_(pre) 705. As shown in FIG. 9, the voltage at theintegrating capacitor (V_(ncap)) jumps back up to the level of V_(pre).As process 800 is repeated a plurality of times, a saw-tooth likevoltage waveform is observed at the integrating capacitor over time.FIG. 9 also illustrates an extrapolation curve 904 showing the RCvoltage decay over time had the voltage V_(pre) 705 not been reasserted.

As shown above, configuring the voltage applied across the nanopore tovary over a time period during which the nanopore is in a particulardetectable state has many advantages. One of the advantages is that theelimination of the operational amplifier that is otherwise fabricatedon-chip in the cell circuitry significantly reduces the footprint of asingle cell in the nanopore based sequencing chip, thereby facilitatingthe scaling of the nanopore based sequencing chip to include more andmore cells (e.g., having millions of cells in a nanopore basedsequencing chip).

Another advantage is that the circuitry of a cell does not suffer fromoffset inaccuracies because V_(pre) is applied directly to the workingelectrode without any intervening circuitry. Another advantage is thatsince no switches are being opened or closed during the measurementintervals, the amount of charge injection is minimized.

Furthermore, the technique described above operates equally well usingpositive voltages or negative voltages. The voltage may be analternating current (AC) voltage. Bidirectional measurements have beenshown to be helpful in characterizing a molecular complex. In addition,bidirectional measurements are valuable when the type of ionic flow thatis driven through the nanopore is via non-faradaic conduction. Two typesof ionic flow can be driven through the nanopore: faradaic conductionand non-faradaic conduction. In faradaic conduction, a chemical reactionoccurs at the surface of the metal electrode. The faradaic current isthe current generated by the reduction or oxidation of some chemicalsubstances at an electrode. The advantage of non-faradaic conduction isthat no chemical reaction happens at the surface of the metal electrode.

FIG. 11 illustrates an embodiment of a time signal plot of a pluralityof signals in a cell of the nanopore based sequencing chip. In someembodiments, the signals are provided by the circuitries shown in FIGS.7A and 7B. The topmost plot of V_(liq) shows the voltage applied tocommon electrode 716 by voltage source V_(liq) 720 over time. The middleplot shows a reset signal 703 that is used to control switch 701 versustime, wherein switch 701 connects or disconnects voltage source V_(pre)705 to or from the membrane in a cell of the nanopore based sequencingchip. The bottom plot shows the voltage at the integrating capacitor(V_(ncap)) in response to the reset signal and V_(liq) as a function oftime.

The topmost plot of V_(liq) shows the voltage applied to the commonelectrode 716 by voltage source V_(liq) 720 over time. Because commonelectrode 716 is immersed in bulk liquid 718, V_(liq) is applied to thebulk liquid in contact with the lipid membranes/bilayers in themeasurement cells. V_(liq) is also referred to as the liquid voltage. Insome embodiments, V_(liq) is a square wave with a peak-to-peak magnitudevariation of 200-250 mV and a frequency between 20 and 400 Hz (e.g., 40Hz). For example, V_(liq) may switch between the values of 0.8 V and 1.0V. The time period when V_(liq) is at the lower value (e.g., 0.8 V) isreferred to as the bright period or tag-reading period, because it isthe period when a tag may be pulled into the nanopore for analysis. Thelower voltage value is also referred to as the tag-reading voltage. Thetime period when V_(liq) is at the higher value (e.g., 1.0 V) isreferred to as the dark period or open-channel period, because it is theperiod during which a tag-attached polyphosphate is repelled and absentfrom the barrel of the nanopore. The higher voltage value is alsoreferred to as the open-channel voltage.

The middle plot shows a reset signal 703 that is used to control switch701 versus time, wherein switch 701 connects or disconnects voltagesource V_(pre) 705 to or from the membrane (also n_(cap) 708) in a cellof the nanopore based sequencing chip. When the reset signal is held athigh during the time periods t_(pre-charging), switch 701 is closed, andwhen the reset signal is held at low during the time periodst_(integrating), switch 701 is open. One t_(pre-charging) periodcombined with one t_(integrating) period together form a single frame.In some embodiments, the frame rate is 2 kHz with a single frameduration of 500 μs. In other embodiments, the frame rate is within therange of 200 Hz to 15 kHz. For ease of illustration, only three framesof the reset signal are drawn within a single bright period of V_(liq)in FIG. 11. However, it should be recognized that there may be tens orhundreds of frames within each bright period or dark period.

When switch 701 is closed during a time period t_(pre-charging), voltagesource V_(pre) 705 is connected to working electrode 714, applying avoltage V_(pre) to the cell working electrode, and the capacitorassociated with the membrane (C_(bilayer) 726) and integrating capacitorn_(cap) 708 are pre-charged to the voltage V_(pre). Accordingly, thevoltage at the integrating capacitor, V_(ncap) (see the bottom plot ofFIG. 11), is equal to V_(pre) within each time period t_(pre-charging).In some embodiments, voltage source V_(pre) 705 provides a constantpositive voltage with a magnitude of 0.9 V. In other embodiments,V_(pre) is a constant voltage selected from within a range of 0.7 to 1.2V.

After the capacitors are pre-charged, switch 701 is opened by the lowreset signal during a time period t_(integrating), decoupling thenanopore, the membrane, and n_(cap) 708 from voltage source V_(pre) 705.During a time period t_(integrating), the capacitors either discharge orcharge, and V_(ncap) either decays or increases exponentially, dependingon the level of V_(liq). When the time period t_(integrating) fallswithin a bright period of V_(liq), V_(liq) is at a lower voltage levelthan V_(ncap), and this potential difference causes the capacitors todischarge and V_(ncap) to decay exponentially. When the time periodt_(integrating) falls within a dark period of V_(liq), V_(ncap) is at alower voltage level than V_(liq), and this potential difference causesthe capacitors to charge up and V_(ncap) to increase exponentiallyinstead. The exponential voltage change has a RC time constant τ=RC,where R is the resistance associated with the nanopore (R_(bilayer) 728)and C is the capacitance in parallel with R, including the capacitanceassociated with the membrane C_(bilayer) 726 and the capacitanceassociated with n_(cap) 708. It should be recognized that the steepnessof the slope of V_(ncap) is not drawn to scale in FIG. 11. The rate ofthe decay in V_(ncap) caused by the discharging of the capacitors mayvary from frame to frame, and the different rates of decay may be usedto analyze a molecule inside a nanopore. The rate of the increase inV_(ncap) caused by the charging of the capacitors may also vary fromframe to frame, and the different rates of increase in V_(ncap) may bemonitored for calibrating the open-channel state, such as driftcorrection.

In the embodiment shown in FIG. 11, the reset signal is kept high for abrief (0.5 to 50 microseconds) t_(pre-charging) period only, and as soonas the capacitors are pre-charged to V_(pre), the reset signal is set toa low level to open the switch, allowing the voltage at integratingcapacitor ncap 708 to float and change to a voltage other than V_(pre).Quick transitions of the reset signal, especially the quick transitionsof the reset signal when V_(liq) switches from a dark period to a brightperiod or vice versa, have been found to create certain V_(ncap) timeprofiles that may cause a number of performance issues in the nanoporebased sequencing chip. For example, the quick transitions of the resetsignal and the corresponding V_(ncap) time profile may causeunpredictable transient characteristics in the lipid bilayer, affectingthe measurement of the rate of change of V_(ncap) during the integratingperiods. The quick transitions of the reset signal also reduce thethreading probability, i.e., the probability of a tag being captured inthe barrel of a nanopore during the bright periods. Therefore, improvedreset signal patterns and V_(ncap) voltage patterns would be desirable.

FIG. 12 illustrates another embodiment of a reset signal that is used tocontrol the switch that connects or disconnects the voltage source to orfrom the membrane in a cell of the nanopore based sequencing chip, suchthat the capacitor associated with the membrane is charged anddischarged repeatedly. In some embodiments, the signals are provided bythe circuitries shown in FIGS. 7A and 7B. The topmost plot V_(liq) isidentical to the V_(liq) plot in FIG. 11. The middle plot shows animproved reset signal 703 that is used to control switch 701 versustime, wherein switch 701 connects or disconnects voltage source V_(pre)705 to or from the membrane in a cell of the nanopore based sequencingchip. The bottom plot shows the voltage at the integrating capacitor(V_(ncap)) in response to the new improved reset signal and V_(liq) as afunction of time.

The topmost plot V_(liq) shows the voltage applied to the commonelectrode 716 by voltage source V_(liq) 720 over time. In someembodiments, V_(liq) is a square wave with a peak-to-peak magnitudevariation of 200-250 mV and a frequency between 20 and 400 Hz (e.g., 40Hz). For example, V_(liq) may switch between the values of 0.8 V and 1.0V. The time period when V_(liq) is at the lower value (e.g., 0.8 V) isreferred to as the bright period or tag-reading period. The time periodwhen V_(liq) is at the higher value (e.g., 1.0 V) is referred to as thedark period or open-channel period.

The middle plot shows an improved reset signal 703 that is used tocontrol switch 701 versus time, wherein switch 701 connects ordisconnects voltage source V_(pre) 705 to or from the membrane (alson_(cap) 708) in a cell of the nanopore based sequencing chip. When thereset signal is held at high during the time periods t_(pre-charging),switch 701 is closed, and when the reset signal is held at low duringthe time periods t_(integrating), switch 701 is open. Onet_(pre-charging) period followed by one t_(integrating) period togetherform a single frame.

In contrast to the frames in FIG. 11, the duration of the frames in FIG.12 are not kept constant over time. There are two types of frames in thereset signal. The different types of frames result in different V_(ncap)patterns, and thus the corresponding improved techniques are referred toas the voltage mode with hybrid mode stimuli. One type of frame (seee.g., frame 1202) is similar to the frames in FIG. 11. In these frames,the reset signal is maintained at a high level for a very brieft_(pre-charging) period only, and as soon as the capacitors arepre-charged to V_(pre), the reset signal is set to a low level to openthe switch, allowing the voltage at integrating capacitor ncap 708 tofloat and change to a voltage other than V_(pre). A second type of frame(see e.g., frame 1204) has longer pre-charging periods than the firsttype of frames. As shown in frame 1204, the reset signal is kept at thehigh level for a longer t_(pre-charging) period. In particular, thereset signal is maintained at the high level during the ending portionof a V_(liq) dark period and the beginning portion of a V_(liq) brightperiod, such that there are no quick transitions of the reset signalwhen V_(liq) switches from a dark period to a bright period.

By extending the pre-charging period in frame 1204, voltage sourceV_(pre) 705 is connected to working electrode 714 for a longer period,and the voltage at the integrating capacitor, V_(ncap) (see the bottomplot of FIG. 12), is maintained at V_(pre) when V_(liq) switches from adark period to a bright period. In some embodiments, voltage sourceV_(pre) 705 provides a constant positive voltage with a magnitude of 0.9V. In other embodiments, V_(pre) is a constant voltage selected fromwithin a range of 0.7 to 1.2 V.

Maintaining V_(ncap) at a constant higher level when V_(liq) switchesfrom a dark period to a bright period has a number of advantages. One ofthe advantages is that it increases the threading probability, theprobability of a tag being captured in the barrel of a nanopore duringthe bright periods. Maintaining V_(ncap) at a constant higher voltage atthe beginning portion of a V_(liq) bright period increases the threadingprobability because the electrical force that can pull a tag into thenanopore remains high for a longer period of time, thereby increasingthe chance that the tag is captured into the nanopore for measurementand decreasing the chance that a tag already trapped in the nanopore mayescape from the nanopore. Another advantage of maintaining V_(ncap) at aconstant higher level when V_(liq) switches from a dark period to abright period is that it reduces the transient characteristics in thelipid bilayer, such as the RC time constant. By having more stabilizedcharacteristics in the lipid bilayer, the measurement of the rate ofchange of V_(ncap) during the integrating periods becomes more reliable.

As shown in FIG. 12, the reset signal is maintained at the high level atboth the ending portion of a V_(liq) dark period and the beginningportion of a V_(liq) bright period. However, in some other embodiments,the reset signal may be maintained at the high level at the endingportion of a V_(liq) dark period only. In some other embodiments, thereset signal may be maintained at the high level at the beginningportion of a V_(liq) bright period only.

In some embodiments, the pre-charging period of frame 1202 isconfigurable. The pre-charging period, t_(pre-charging), of frame 1202is configured to a predetermined duration ranging from 50 microsecondsto 1 millisecond seconds. In some embodiments, the pre-charging periodof frame 1204 is configurable. The pre-charging period,t_(pre-charging), of frame 1204 is configured to a predeterminedduration ranging from 100 microseconds to 50 milliseconds. Thepre-charging period that overlaps with the V_(liq) dark period isbetween 50 microseconds to 25 milliseconds. The pre-charging period thatoverlaps with the V_(liq) bright period is between 50 microseconds to 25milliseconds.

It should be recognized that the steepness of the slope of V_(ncap) isnot drawn to scale in FIG. 12. The rate of decay in V_(ncap) caused bythe discharging of the capacitors may vary from frame to frame, and thedifferent rates of decay may be used to analyze a molecule inside ananopore. The rate of increase in V_(ncap) caused by the charging of thecapacitors may also vary from frame to frame, and the different rates ofincrease in V_(ncap) may be monitored for calibrating the open-channelstate, such as drift correction.

For ease of illustration, only two integration periods are drawn withina single bright period of V_(liq) in FIG. 12. Similarly, only oneintegration period is drawn within a single dark period of V_(liq) inFIG. 12. However, it should be recognized that there may be tens orhundreds of integration periods within each bright period or darkperiods, as illustrated in FIG. 13.

FIG. 14 illustrates yet another embodiment of a reset signal that isused to control the switch that connects or disconnects the voltagesource to or from the membrane in a cell of the nanopore basedsequencing chip, such that the capacitor associated with the membrane ischarged and discharged repeatedly. In some embodiments, the signals areprovided by the circuitries shown in FIGS. 7A and 7B. The topmost plotof V_(liq) is identical to the V_(liq) plots in FIGS. 11 and 12. Themiddle plot shows another improved reset signal 703 that is used tocontrol switch 701 that connects or disconnects voltage source V_(pre)705 to or from the membrane in a cell of the nanopore based sequencingchip. The bottom plot shows the voltage at the integrating capacitor(V_(ncap)) in response to the new improved reset signal and V_(liq) as afunction of time.

In contrast to the frames in FIG. 11, the duration of the frames in FIG.14 are not kept constant over time. There are two types of frames in thereset signal. One type of frame (see e.g., frame 1202) is similar to theframes in FIG. 11. In these frames, the reset signal is maintained at ahigh level for a very brief t_(pre-charging) period only, and as soon asthe capacitors are pre-charged to V_(pre), the reset signal is set to alow level to open the switch, allowing the voltage at integratingcapacitor ncap 708 to float and change to a voltage other than V_(pre).A second type of frame (see e.g., frame 1402) has longer pre-chargingperiods than the first type of frame. As shown in frame 1402, the resetsignal is kept at the high level for a longer t_(pre-charging) period.In particular, the reset signal is maintained at the high level duringthe ending portion of a V_(liq) bright period and the beginning portionof a V_(liq) dark period, such that there are no quick transitions ofthe reset signal when V_(liq) switches from a bright period to a darkperiod.

By extending the pre-charging period in frame 1402, voltage sourceV_(pre) 705 is connected to working electrode 714 for a longer period,and the voltage at the integrating capacitor, V_(ncap), (see the bottomplot of FIG. 14) is maintained at V_(pre) when V_(liq) switches from abright period to a dark period. In some embodiments, voltage sourceV_(pre) 705 provides a constant positive voltage with a magnitude of 0.9V. In other embodiments, V_(pre) is a constant voltage selected fromwithin a range of 0.7 to 1.2 V.

Maintaining V_(ncap) at a constant higher level when V_(liq) switchesfrom a bright period to a dark period has its advantages. One advantageof maintaining V_(ncap) at a constant higher level when V_(liq) switchesfrom a bright period to a dark period is that it reduces the transientcharacteristics in the lipid bilayer, such as the RC time constant. Byhaving more stabilized characteristics in the lipid bilayer, themeasurement of the rate of change of V_(ncap) during the integratingperiods becomes more reliable.

As shown in FIG. 14, the reset signal is maintained at the high level atboth the ending portion of a V_(liq) bright period and the beginningportion of a V_(liq) dark period. However, in some other embodiments,the reset signal may be maintained at the high level at the endingportion of a V_(liq) bright period only. In some other embodiments, thereset signal may be maintained at the high level at the beginningportion of a V_(liq) dark period only.

In some embodiments, the pre-charging period of frame 1202 isconfigurable. The pre-charging period, t_(pre-charging), of frame 1202is configured to a predetermined duration ranging from 50 microsecondsto 1 millisecond seconds. In some embodiments, the pre-charging periodof frame 1402 is configurable. The pre-charging period,t_(pre-charging), of frame 1402 is configured to a predeterminedduration ranging from 100 microseconds to 50 milliseconds. Thepre-charging period that overlaps with the V_(liq) bright period isbetween 50 microseconds to 25 milliseconds. The pre-charging period thatoverlaps with the V_(liq) dark period is between 50 microseconds to 25milliseconds.

In some embodiments, the reset signal includes three types offrames—frames 1202, frames 1204, and frames 1402. By having both frames1204 and 1402 in the reset signal, the voltage at the integratingcapacitor, V_(ncap), is maintained at V_(pre) when V_(liq) switches froma bright period to a dark period or vice versa.

Although the foregoing embodiments have been described in some detailfor purposes of clarity of understanding, the invention is not limitedto the details provided. There are many alternative ways of implementingthe invention. The disclosed embodiments are illustrative and notrestrictive.

All publications, patents, patent applications, and/or other documentscited in this application are incorporated by reference in theirentirety for all purposes to the same extent as if each individualpublication, patent, patent application, and/or other document wereindividually indicated to be incorporated by reference for all purposes.

1. A method of analyzing a molecule in a nanopore, comprising: applyinga liquid voltage to an electrolyte on a first side of a lipid bilayer,wherein the liquid voltage comprises a tag-reading period with atag-reading voltage level that tends to capture a tag of the molecule ora portion of the molecule into a nanopore in the lipid bilayer and anopen-channel period with an open-channel voltage level that tends torepel the tag or the portion of the molecule from the nanopore in thelipid bilayer; connecting a pre-charging voltage source to anintegrating capacitor and a working electrode on a second side of thelipid bilayer during a pre-charging time period, such that theintegrating capacitor and the working electrode are charged to apre-charging voltage; and disconnecting the pre-charging voltage sourcefrom the integrating capacitor and the working electrode during anintegrating time period, such that a voltage of the integratingcapacitor and a voltage of the working electrode may vary as a currentflows through the nanopore in the lipid bilayer; wherein thepre-charging time period overlaps with a beginning portion of thetag-reading period.
 2. The method of claim 1, wherein the pre-chargingtime period overlaps with an ending portion of the open-channel period.3. The method of claim 1, wherein the pre-charging time period spansacross a transition from the open channel period to the tag-readingperiod.
 4. The method of claim 1, wherein the pre-charging time periodoverlaps with a beginning portion of the open-channel period.
 5. Themethod of claim 1, wherein the pre-charging time period overlaps with anending portion of the tag-reading period.
 6. The method of claim 1,wherein the pre-charging time period spans across a transition from thetag-reading period to the open-channel period.
 7. The method of claim 3,wherein the pre-charging time period has a duration of 100 microsecondsto 50 milliseconds.
 8. The method of claim 1, wherein the pre-chargingtime period has a duration of 50 microseconds to 25 milliseconds.
 9. Asystem, instrument or device for analyzing a molecule, comprising: avoltage source; a counter electrode coupled to the voltage source; apre-charging voltage source; an integrating capacitor that isconfigurable to connect with the pre-charging voltage source; a workingelectrode that is configurable to connect with the pre-charging voltagesource; and a controller configured to: control the voltage source toapply a liquid voltage via the counter electrode to an electrolyte on afirst side of a lipid bilayer, wherein the liquid voltage comprises atag-reading period with a tag-reading voltage level that tends tocapture a tag of the molecule or a portion of the molecule into ananopore in the lipid bilayer and an open-channel period with anopen-channel voltage level that tends to repel the tag or the portion ofthe molecule from the nanopore in the lipid bilayer; connect thepre-charging voltage source to the integrating capacitor and the workingelectrode on a second side of the lipid bilayer during a pre-chargingtime period, such that the integrating capacitor and the workingelectrode are charged to a pre-charging voltage; disconnect thepre-charging voltage source from the integrating capacitor and theworking electrode during an integrating time period, such that a voltageof the integrating capacitor and a voltage of the working electrode mayvary as a current flows through the nanopore in the lipid bilayer; andwherein the pre-charging time period overlaps with a beginning portionof the tag-reading period; and a memory coupled to the controller andconfigured to provide the controller with instructions.
 10. The system,instrument or device of claim 9, wherein the pre-charging time periodoverlaps with an ending portion of the open-channel period.
 11. Thesystem, instrument or device of claim 9, wherein the pre-charging timeperiod spans across a transition from the open channel period to thetag-reading period.
 12. The system, instrument or device of claim 9,wherein the pre-charging time period overlaps with a beginning portionof the open-channel period.
 13. The system, instrument or device ofclaim 9, wherein the pre-charging time period overlaps with an endingportion of the tag-reading period.
 14. The system, instrument or deviceof claim 9, wherein the pre-charging time period spans across atransition from the tag-reading period to the open-channel period. 15.The system, instrument or device of claim 9, wherein the pre-chargingtime period has a duration of 100 microseconds to 25 milliseconds.